Papillomaviruses induce a variety of skin lesions which are normally benign, but occasionally progress to malignant carcinomas such as cervical cancer. The E1 and E2 open reading frames of the papillomaviruses encode multiple proteins that regulate viral gene expression and DNA replication. The E1 and E2 proteins have both positive and negative effects on the viral life cycle and so a detailed understanding of their regulatory mechanisms is crucial for the design of antiviral drugs and strategies. In the past year we have continued to characterize the structure and function of the E1 and E2 gene products. We have further characterized nuclear localization signals in the E2 transactivator and repressor proteins and have preliminary evidence that the localization of the E2 transactivator protein changes during the cell-cycle. This differential localization may define a new function for the E2 proteins in segregating viral genomes at cell division. We have also continued to analyze how the functions of the E2 proteins are regulated by phosphorylation and are developing novel methods for isolating cellular proteins that interact with the viral E2 proteins. The full-length E1 gene product is the predominant protein required for viral DNA replication but it also functions as a transcriptional repressor. We have previously defined the domains of the E1 protein required for sequence-specific origin binding and interaction with the E1 protein. We have now extended these studies and have determined that the functions and domains required for DNA replication and transcriptional repression are separable.